The initial event of mammalian fertilization is the interaction of fertilization-competent male and female gametes (the sperm and egg, respectively). During spermatogenesis and epididymal maturation, the plasma membrane of the sperm becomes capable of binding to the extracellular matrix of the egg, the zona pellucida. This sperm-egg interaction is species-specific and leads to the induction of the acrosome reaction which is ultimately required for successful fertilization. These interactions have many characteristics of ligand-receptor-effector interactions which regulate numerous somatic cell functions. While the zona pellucida component that binds to the sperm plasma membrane has been shown to be an egg-specific glycoprotein called ZP3, the receptor on the sperm membrane remains unknown. A number of candidate ZP3 receptors have been suggested including a major phosphotyrosine-containing protein of Mr 95,000. Recently, this protein has been purified and shown to be a unique membrane-bound, tyrosine-phosphorylated form of the glycolytic enzyme hexokinase. Results of cloning studies suggest that sperm may contain multiple forms of hexokinase, some of which are similar to the somatic forms and some of which appear to be unique to spermatogenic cells. The long-term objective of this-proposal is to examine whether this hexokinase in sperm has a role in sperm-egg interaction. The specific aims are: 1) to determine which isoform(s) of hexokinase is present in mature sperm and its location; specifically, whether any isoform localizes to the sperm membrane overlying the acrosome, the site of sperm-egg interaction, 2) to determine which isoform(s) of hexokinase is phosphorylated on tyrosine residues in the mature sperm and the mechanism by which it becomes phosphorylated, i.e. an autophosphorylation event and/or by the action of a tyrosine kinase, and 3) to determine whether hexokinase in mature sperm participates in the sperm-zona pellucida interaction; specifically, can this interaction be inhibited with probes that interfere with hexokinase. In relation to health the results from these experiments will promote a deeper understanding of sperm-egg binding and interaction. Moreover, these experiments may lead to new approaches to contraception and to a comprehension of specific forms of infertility.